Nucleic acid hybridization is a widely used method for identifying specific sequences of nucleic acids. Hybridization is based upon pairing between complementary nucleic acid strands. Single-stranded oligonucleotides having known sequences can be used as hybridization probes to identify target sequences of nucleic acid analytes, by exposing the probes to sample solutions containing a nucleic acid analyte of interest. If a nucleic acid analyte hybridizes to a probe, the analyte necessarily contains the target sequence. Various aspects of this method have been studied in detail. In essence, all variations allow complementary base sequences to pair and form double-stranded molecules, and a number of methods are known in the art to determine whether pairing has occurred, such as those described in U.S. Pat. No. 5,622,822 to Ekeze et al. and U.S. Pat. No. 5,256,535 to Ylikoski et al.
Another method of detecting the binding of a probe to a target sequence is described by Whitcombe et al. (1999), “Detection of PCR products using self-probing amplicons and fluorescence,” Nature Biotechnology 17:804-807. The method involves use of a probe containing a nucleotide sequence that is not amplified during the polymerase chain reaction (PCR), wherein the probe is designed to form a hairpin structure in which a fluorophore and a quencher are in self-quenching proximity. Upon denaturation and hybridization with a target nucleotide sequence, however, the segment of the probe that was formerly in the hairpin structure hybridizes with the amplified target sequence, and is detectable by the increased fluorescence. This “Scorpion” probe combines the functions of a PCR primer and a detection probe and provides enhanced amplification and detection, in part due to the unimolecular nature of the binding reaction of the fluorescent probe to the target nucleotide sequence, which increases the rate and extent of the reaction. This technology is also described in U.S. Pat. No. 5,525,494 to Newton. Detection of genetic variation among individuals is another application of sequence-specific hybridization technologies. Detection and analysis of allelic variations or SNPs in DNA typically involves chain extension and amplification using primers targeted for a specific sequence. The amplified DNA is then used as a target for various labeled oligonucleotide probes to identify point mutations and allelic sequence variation. If, however, the target DNA forms intra-molecular secondary structures, the DNA may not be able to hybridize with the primer or labeling probes efficiently or at all, thus resulting in no signal for the presence or absence of an SNP at the location of the secondary structure.
Such intramolecular secondary structures in a single-stranded nucleic acid, such as rRNA or denatured DNA, arise from the intramolecular formation of hydrogen bonds between complementary nucleotide sequences within the single-stranded nucleic acid itself. This residual secondary structure can sterically inhibit, or even block, hybrid formation between an oligonucleotide, for example a DNA or RNA oligomer being used as a probe, and its complementary sequence in the RNA or DNA (e.g., ribosomal RNA, mRNA, or DNA) that the probe targets.
There are numerous cases in which there is difficulty in determining the presence or absence of SNPs in a particular target nucleic acid. For example, cytochrome P450 exists in several allelic variations, which are associated with altered metabolism of drugs and/or cancer susceptibility. One variant of cytochrome P450, cytochrome P450 CYP2D6, has an SNP in each of the four exons of the P450 CYP2D6 gene. In experiments performed to test for the presence of these SNPs, difficulties have been encountered in detecting SNPs in exons 1 and 2, due to significant secondary structure in the regions of analytical interest in these exons. Although exons 1 and 2 can be amplified with conventional primer sets, the product cannot be detected in the SNP assay. Problems in probing exons 1 and 2 were eventually linked to intra-molecular secondary structure in these two amplicons. This secondary structure precludes hybridization of allele-specific discrimination probes that would be used in an assay for these SNPs. The structures of these exons are shown in FIG. 1, where it can be seen that exons 6 and 9 exhibit relatively little difficulty for PCR and hybridization with discrimination probes, whereas exons 1 and 2 show significant secondary structure in the region of the SNP. The analysis region and the SNP are shown in red in FIG. 1.
One solution to this problem of enhancing hybridization of a probe to a target nucleotide sequence when the target sequence forms intramolecular secondary structures has been proposed in U.S. Pat. No. 5,030,557 to Hogan et al. In this patent, Hogan et al. describe how the addition of a helper nucleic acid sequence in molar excess (at least about 5 times that of the probe and up to 100 or more times that of the probe) to the nucleic acid probe sequence aids in hybridization of the probe sequence to the target sequence. In fact, Hogan et al. demonstrate the effectiveness of their approach using molar ratios of helper oligonucleotide to probe oligonucleotide of 60:1, 100:1 and 250:1. In addition, Hogan et al. state that the helper oligonucleotides should be longer than about 20 to about 50 nucleotides in length in order to be effective in blocking the formation of the secondary structure.
Therefore, there is a need in the art to provide improved hybridization probes and methods of detecting sequences contained within regions of a target molecule that tend to form an unwanted secondary structure. The method described by Hogan et al. provides one such solution. This method, however, has the disadvantages of requiring a large excess of helper oligonucleotide over probe oligonucleotide, and of constraining the length of the helper oligonucleotides.